1. Field of the Invention
The present invention provides medical research and diagnostic methods and reagents for detecting and typing HPV.
The methods utilize PCR, a DNA amplification technique widely used in the fields of molecular biology and genetic engineering. The methods can also be used to generate information concerning previously unknown types and sub-types of HPV and consequently has applications in the field of virology.
2. Description of Related Art
Papillomaviruses have been linked to widespread, serious human diseases, especially carcinomas of the genital and oral mucosa. And although genital HPV infection is associated with cancer primarily in women, recent evidence suggests that HPV may play a role in the development of anogenital cancers in men. Broker et al., 1986, Cancer Cells 44:17-36, review the molecular, cellular, and clinical aspects of the papillomaviruses and the relationship of HPVs to cancer. HPV types 6, 11, 16, 18, and 33 are known genital HPV types in the human population, and Broker et al., 1986, Cancer Cells 4:589-594, disclose that HPV types 6, 11, 16, 18, and 33 share significant homology at the DNA level, particularly at the L1 open reading frame.
Identification and typing of HPV is quite important, because different types of HPV pose different risks to the affected individuals. For instance, HPV 16 and HPV 18 have been more consistently identified in higher grades of cervical dysplasia and carcinoma than other HPV types. Webb et al., December 1987, J. Inf. Disease 156(6):912-919, report a method for detecting HPV DNA types that utilizes a reverse-blotting procedure. The procedure involved forming a membrane to which genomic DNA from four different HPV types was bound and then hybridizing labeled DNA from a biological sample to the DNA bound to the membrane. Caussey et all., February 1988, J. Clin. Microbiol. 26(2):236-243 describe similar HPV detection methods.
Shibata et al., Jan. 1988, J. Exp. Med. 167:225-230, disclose the use of PCR to amplify and detect the presence of HPV16 and HPV18 DNA. U.S. Pat. Nos. 4,683,195 and 4,683,202 disclose PCR and the use of PCR to detect the presence or absence of nucleic acid sequence in a sample. PCT Patent Publication No. WO 90/02821 discloses the use of consensus primers and probes to amplify and detect HPV sequences in a sample. The publication also describes type-specific probes for typing the HPV DNA if present in the sample.
Maitland et at., May 1988, Seventh International Papillomavirus Workshop, Abstract, p. 5, report the use of PCR to detect HPV16 in oral and cervical biopsies. In addition, Campione-Piccardo et al., May 1988, Seventh International Papillomavirus Workshop, Abstract, p. 19, report the use of a mixture of primers for the specific amplification by PCR of HPV sequences in types 1a, 5, 6a, 6b, 8, 11, 16, 18, and 33. A number of other researchers disclosed the use of PCR to amplify and detect HPV sequences at the Seventh International Papillomavirus Workshop. Each of the background references described in this section is incorporated herein by reference.
The heterogeneity of the human papillomavirus group is generally described in deVilliers, 1989, J. Virology 63:4898-4903, which is incorporated herein by reference. The genomes of numerous HPV types have been sequenced and/or characterized. For example, for HPV type 6, see de Villiers et al., 1981, J. Virology 40:932-935, and Gissmann and Zur Hausen, 1980, Int. J. Cancer 25:605-609, and Schwartz et al., 1983, EMBO J. 2(12):2341-2348. For HPV type 2, see Gissmann et al., 1982, J. Virology 44:393-400. For HPV type 11, see Dartmann et al., 1986, Virology 151:124-130. For HPV type 16, see Seedorf et al., 1985, Virology 145:181-185. For HPV type 18, see Cole and Danos, 1987, J. Mol. Biol. 193:599-608. For HPV type 31, see Goldsborough et al., 1989, Virology 171:306-311. For HPV 33, see Cole and Streeck, 1986, J. Virology 58:991-995. For HPV 54, see Favre et al., 1990, Int. J. Cancer 45:40-46. For HPV 56, see Lorincz, 1989, J. Gem Virol. 70:3099. These publications are incorporated herein by reference.
Despite the use of PCR to amplify and detect HPV sequences, there still remains a need for a simple and rapid method for both detecting and typing HPV in a biological sample. Patent Publication No. PCT/US89/03747 describes improved methods that offer speed and simplicity for detection and typing HPV in a sample. The methods comprise amplifying a sequence of HPV DNA present in the sample, determining if amplification has occurred and then hybridizing an HPV type-specific probe to the amplified DNA.